P835: IN SILICO PREDICTION REVEALS PUTATIVE T-CELL CLASS I/II NEOEPITOPES WITHIN THE CLONOTYPIC IMMUNOGLOBULIN HEAVY AND LIGHT CHAINS IN PATIENTS WITH MULTIPLE MYELOMA
Authors: Glykeria Gkoliou, Nikos Pechlivanis, Sofia Chatzileontiadou, Charikleia Xydopoulou, Christina Frouzaki, Georgios Karakatsoulis, Elisavet Vlachonikola, Marina Gerousi, Fotis Psomopoulos, Alexandra Siorenta, Maria Papaioannou, Katerina Chlichlia, Kostas Stamatopoulos, Evdoxia Hatjiharissi, Anastasia Chatzidimitriou
Journal: HemaSphere (2023)

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: Several T cell defects have been identified in multiple myeloma (MM), particularly pertaining to T cell exhaustion and senescence, likely as a result of chronic antigenic stimulation. Arguably, therefore, antigenic stimulation is highly relevant for shaping the T cell repertoire in MM, however the nature of the implicated antigens remains elusive. Similar to other mature B cell malignancies, immunoglobulin (IG) gene rearrangements in MM lead to the expression of unique, novel peptide sequences that can serve as neoantigens for T cells. Aims: We performed ad hoc prediction of putative T-cell class I/II neoepitopes contained within the MM clonotypic IG gene rearrangements. Methods: We studied 25 newly diagnosed patients with MM from whom we isolated the peripheral blood and/or bone marrow mononuclear cell fraction. Clonotypic IG heavy and light chain gene rearrangements were RT-PCR amplified on RNA extracted from the CD138+ myeloma cells using subgroup-specific leader primers for the IGHV/IGKV/IGLV gene and universal primers annealing to the constant domain. The corresponding sequences were determined by bidirectional Sanger sequencing. T cell receptor beta (TRB) chain gene rearrangements were RT-PCR amplified on RNA extracted from CD4+ and CD8+ T cells and then subjected to paired-end NGS. The deduced amino acid sequences of the IG heavy and light chains were subjected to bioinformatics analysis for the identification of putative T-cell class I/II neoepitopes using the NetMHCpan and NetMHCIIpan softwares. High- and medium-binding peptides (rank score <2%) were selected, considering the 4-digit HLA-A, -B, -C and DRB1 typing for each individual patient. Prediction of the binding specificity of T Cell Receptors (TR) to MHC-peptide complexes (pMHCs) was performed by the ERGO-II tool. The rank score was calculated, considering the peptides resulted by NetMHCpan/NetMHCIIpan, the HLA and the clonotypes for each patient (AUC>0.8). Exact matches to germline and/or proteome databases were excluded. Results: All patients displayed oligoclonal T cell expansions in both CD4+ and CD8+ T cells, albeit these were significantly (p<0.001) more pronounced in the latter. Both CD4+ and CD8+ T cell subpopulations displayed skewed TRBV and TRBJ gene repertoires (7 TRBV genes and 3 TRBJ genes accounting for about 40% and 59% of the repertoire, respectively). Overall, 821 predicted neoepitopes were identified. All patients had predicted CD4+ and CD8 + T-cell epitopes within the MM clonotypic IG, either in the heavy chain (25/25 pts, n=420 epitopes) or the light chain (25/25 pts, n=401 epitopes). Interestingly, 740/821 (90%) peptides with strong binding affinity resulted from IG gene sequence motifs outside the VH/VK/VL CDR3. Most of these peptides (618/821, 75%) resulted from somatic hypermutations (SHM) across the IG variable domain. Overall, 64,993 clonotypes of CD4+ T cells and 14,695 clonotypes of CD8+ T cells were in silico predicted to have strong binding affinity. Importantly, 1697 clonotypes with high binding score were found to be shared between CD4+ T cells of all patients and 222 clonotypes between CD8+ T cells of all patients and, thus, were deemed as public. Summary/Conclusion: The clonotypic IG of MM patients contains a significant number of putative T-cell class I/II neoepitopes, most of which derive from SHM. This implies that the SHM which shapes the MM BcR IG repertoire may produce immunogenic CD4+/CD8+ T cell epitopes. Their actual immunogenicity has to be tested in ex vivo studies, currently underway by our group. Keywords: Antigen-specific T cells, Immunoglobulin, Multiple myeloma, T cell